In recent years, genetic information of many organisms, such as human genome, has been decoded. Under the circumstances, functional analysis of proteins and creation of genomic medicine based on such genetic information have been attracting attention for postgenomic studies. Application and utilization of proteins corresponding to such genetic information for pharmaceutical products and the like requires easy synthesis of extensive kinds of proteins in a short time.
At present, expression systems using viable cells (hereinafter sometimes to be referred to as “cell-system”) of yeast, insect cell and the like by the gene recombination technique have been widely utilized as the production methods of proteins. However, viable cells show a propensity toward elimination of exogenous proteins for their functional retention, and there are many proteins that cannot be expressed easily since expression of cytotoxic proteins in viable cells prevents cell growth.
On the other hand, as a production method of protein free of a cell-system, cell-free protein synthesis has been known, which includes adding a substrate, an enzyme and the like to a cell rupture, extract solution and the like to provide a wide choice of genetic information translation systems of organisms in test tubes, and reconstructing a synthetic system capable of linking the necessary number of amino acid residues in a desired order using an mRNA encoding an object protein. Such a cell-free protein synthesis is relatively free of the limitation imposed on the above-mentioned cell-system protein synthesis, and is capable of synthesizing proteins without killing the organism. In addition, because the production of protein does not accompany operations of culture and the like, the protein can be synthesized in a short time as compared to cell-systems. Moreover, inasmuch as the cell-free protein synthesis also affords a large scale production of proteins consisting of amino acid sequences not utilized by the organism, it is expected to be a promising expression method. As a cell rupture or extract solution to be applied to the cell-free protein synthesis, use of various substances of biological derivation has been considered and investigations are underway. Of these, since yeast can be easily cultured like prokaryotes such as Escherichia coli and the like, its extract solution can be obtained at a low cost. Since yeast is an eukaryote, posttranslational modification such as glycosylation and the like, which is not applicable to extract solutions of Escherichia coli and the like, can be applied. In view of the above, the development of yeast-derived extract solutions for cell-free protein synthesis has been drawing much attention.
A cell-free protein synthesis method using a yeast-derived extract solution was first reported by Gasior et al. (for example, Gasior, E. et al., J. Biol. Chem., 254, 3965-3969, 1979). According to the method of Gasior et al., yeast is first cultured, spheroplast is prepared using glusulase and then cultured again in YM-5 medium containing 0.4 M MgSO4. Then cells are recovered again by centrifugation, suspended in a buffer and ruptured with a Dounce homogenizer. The rupture is subjected to centrifugation at 30,000xg, then at 100,000xg. The obtained supernatant is applied to Sephadex G-25 and the fractions having a high protein content are collected to give an extract solution for cell-free protein synthesis. However, the method of Gasior et al. requires very complicated preparation of an extract solution, requiring considerable time and labor therefor.
To solve this problem, Hussain et al. developed a more convenient preparation method of a extract solution for cell-free protein synthesis, by changing the method of cell rupture (for example, Hussain, I et al., Gene, 46, 13-23, 1986). The method proposed by Hussain et al., which is a highly convenient production method of an extract solution, comprises culturing yeast, collecting cells, washing the cells with a buffer, suspending and then rupturing the cells with glass beads, centrifuging the obtained rupture at 30,000xg and subjecting the obtained supernatant to Sephadex G-25.
In addition, US2002/0168705 A1 (JP-A-2002-262867) discloses a cell-free synthesis method of a heavy atom isomorphous replacement product protein, which is suitable for X-ray crystallographic analysis of a protein using an extract solution derived from yeast and the like.
However, the amount of protein synthesized using the extract solution obtained by any of the above-mentioned methods is extremely small, and the protein synthesis activity can be measured only by the uptake of the radiation-labeled amino acid. Therefore, a method for preparing a yeast-derived extract solution, which is easily prepared and capable of synthesizing a high amount of protein, has been desired.